The broad objectives of this research program are the acquisition of a better understanding of the detailed enzymatic mechanisms involved in the transformation of steroid hormones in microbial and mammalian systems. The principal reactions to be studied are: (a) The delta5-3-ketosteroid isomerase reaction which plays a critical role in the biosynthesis of steroid hormones, and (b) Steroidoxidations by nicotinamide adenine dinucleotide-linked hydroxysteroid dehydrogenases. Specific attention will be accorded to: 1. The basic mechanisms of the steroid transformation including the purification of the enzymes involved, their molecular properties, cofactor requirements and catalytic mechanisms. 2. Development of procedures of affinity chromatography for the purifcation of these enzymes and for the analysis of their catalytic processes. 3. Description of substrate and inhibitor specificities of steroid transforming enzymes. Particular effort will be devoted to the study of active-site-directed and suicide substrate inhibitors enzymes. 4. Evaluation of these inhibitors in purified steroid-metabolizing enzyme preparations as well as in more highly organized systems with a view of perturbing normal or abnormal endocrine function. 5. Examination of the properties of isofunctional enzymes in different species, tissues and intra-cellular compartments. 6. The complete description of the details of the active site and mechanism of the crystalline delta5-3-ketosteroid isomerase by affinity labeling, chemical modification, conformational probes, and X-ray crystallography. 7. Development of methods for the enzymatic microestimation of steroid hormones and their metabolites in subpicomole amounts.